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Hindawi Publishing CorporationJournal of NanomaterialsVolume 2012, Article ID 714134, 11 pagesdoi:10.1155/2012/714134

Review Article

Nanotechnology-Based Therapies for Skin Wound Regeneration

Ilaria Tocco,1 Barbara Zavan,2 Franco Bassetto,1 and Vincenzo Vindigni1

1 Institute of Plastic Surgery, University Hospital of Padova, 35128 Padova, Italy2 Department of Histology, Microbiology and Biomedical Technologies, University of Padova, 35128 Padova, Italy

Correspondence should be addressed to Ilaria Tocco, [email protected]

Received 8 February 2012; Revised 19 February 2012; Accepted 24 February 2012

Academic Editor: Xiaoming Li

Copyright © 2012 Ilaria Tocco et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The cutting-edge combination of nanotechnology with medicine offers the unprecedented opportunity to create materials and de-vices at a nanoscale level, holding the potential to revolutionize currently available macroscale therapeutics. Nanotechnologyalready provides a plethora of advantages to medical care, and the success of nanoparticulate systems suggests that a progressiveincrease in the exploration of their potential will take place in the near future. An overview on the current applications of nanotech-nology to wound healing and wound care is presented.

1. Introduction

Nobel Prize winner Richard Feynman was the first in 1959 topredict the future emergence of a new science which could beable to deal with structures on a scale 1–100 nm. Fifty yearsafter, the unpredictable result is the ability to manipulatematerials on the same unimaginably small scale which isused by nature [1]. The repercussions that nanotechnology ishaving in our lives nowadays are towering: the current appli-cations to molecular biology are leading to the developmentof structures, devices, and systems bearing a capacity to re-volutionize medical therapeutics and diagnostics, which hasnever been seen before.

Nanodevices are innovative and can provide a wide rangeof advantages: from the ability of nanoparticles to enter intothe cytoplasmic space across cellular barriers, like Trojanhorses, and activate specific transport mechanisms [2, 3]; tothe modulation of drugs biocompatibility, bioavailability andsafety profiles through nanodelivery systems [4]. Further-more, therapeutic selection can increasingly be tailored toeach patient’s profile. Nanotherapies represent a great oppor-tunity to enhance currently available medical treatments, im-proving standard care and prognosis for challenging health-care issues, like impaired wound healing.

The public health impact of chronic wounds is stagger-ing. An estimated 1.3 to 3 million US individuals are be-lieved to have pressure ulcers and as many as 10% to 15% ofthe 20 million individuals with diabetes are at risk of

developing diabetic ulcers [5]. Nonhealing wounds are theresults of a stop in the progression of the normal sequenceof cellular and biochemical events towards the restoration ofthe skin’s integrity. Factors delaying wound healing includeeither preexisting comorbidities (diabetes, chronic periph-eral vasculopathy, and immunosuppression), leading to lackof appropriate metabolism and clearance from toxic sub-stances of the wound, and/or sudden complications, such asinfections, exalting the inflammatory state. Nanobiotechnol-ogy combined with the knowledge on cellular and subcellulardysfunctional events occurring in delayed wound healingoffers great opportunities for improving wound care.

This paper will focus on the currently available nanotech-nology-based therapies in the field of wound healing and skinregeneration. To appreciate the importance of the functionsof the innovative nanosystems, an understanding of thewound healing process is essential.

1.1. The Wound Healing Process. The wound healing processis classically defined as a series of continuous, sometimesoverlapping, events. These are haemostasis, inflammation,proliferation, epithelisation, maturation, and remodelling ofthe scar tissue [6] (Figure 1).

Haemostatic events occur immediately after injury. Theexposure of subendothelial collagen and the formation ofthrombin lead to the activation of the platelets, located in

2 Journal of Nanomaterials

PDGF; FGF;

es

Haemostasis

VasoconstrictionPlatelet aggregation and degranulationBlood clotting

Inflammation

Leucocyte migration

Removal of cellular debris by macrophages

Release of growth factors

Proliferation

NeovascularisationFibroblasts proliferation and secretion of glycoprotein and collagen

Granulation tissue

Remodelling

Wound remodellingWound contraction

Scar tissue

Wounding

Growthfactors

PDGF; EGF; FGF; TGF-β;TNF

EGF; VEGFes

PDGF; FGF; EGF

MMPs

Neutrophils, secretion of chemicals killing bacteria

Keratinocytes migration from the wound edge

Fibroblasts secretion of collagen and matrix proteins to strengthen wound

TGF-βW

-3 d

ays

3 da

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onth

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3–12

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Figure 1: The main biological phases in wound healing. The events in wound healing and the soluble factors involved in each phase arewell defined. Although presented as separated for absolute clarity, no phase is initiated exactly at the complexion of the previous one, and allphases overlap to a certain degree (see arrows on the left).

the intravascular space. Activated platelets play a trigger rolein a number of events:

(i) activation of the coagulation cascade. This eventuallyleads to the formation of a fibrin clot that acts asscaffolding for other cells that later enter the wound;

(ii) activation of the complement system;

(iii) platelet degranulation: cells release an array of cyto-kines, growth factors, and vasoactive substances fromthe platelet α-granules, such as platelet-derivedgrowth factor (PDGF), transforming growth factor-β(TGF-β), fibroblast growth factor (FGF), endothelialgrowth factor (EGF), platelet-derived angiogene-sis factor, serotonin, bradykinin, platelet-activatingfactor, thromboxane A2, platelet factor IV, prost-aglandins, and histamine. These platelet releasatesinitiate the early events of wound healing [6, 7].

The inflammatory phase begins immediately after injuryand may continue for up to 6 days [8]. Growth factors re-leased from the platelets diffuse into tissues surrounding thewound and chemotactically draw inflammatory cells into the

injured area. Neutrophils are the first inflammatory cells toenter the wound, followed by monocytes. Once chemotaxisis completed, local mediators activate the inflammatory cells.Activated neutrophils release a number of lysosomal enzymes(such as elastase, neutral proteases, and collagenase) whichproteolytically remove damaged components of extracel-lular matrix (ECM) [8]. Activated monocytes acquire themacrophage phenotype and aid in host defence [8].

The proliferation phase is characterised by the formationof the ECM and the beginning of angiogenesis. The primarycells involved in this phase are fibroblasts and endothelialcells. They proliferate in response to growth factors andcytokines that are released from macrophages, platelets andmesenchymal cells, or have been stored in the fibrin clot.In addition to chemotactically drawing fibroblasts into thewound, PDGF, FGF, and EGF induce fibroblast activationand proliferation [9]. During the first 2-3 days after-injury,fibroblasts activity predominantly involves migration andproliferation. After this time, fibroblasts release collagen andglycosaminoglycans (mainly hyaluronic acid, chondroitin-4-sulphate, dermatan sulphate, and heparin sulphate) in

Journal of Nanomaterials 3

response to macrophage-released growth factors, hypoxiaand by-products of anaerobic metabolism. The combinationof collagen and fibronectin forms the new ECM, which is es-sential for the development of granulation tissue that even-tually fills the wound [6]. Angiogenesis accompanies fibrob-last proliferation and allows nutrients and healing factors toenter the wound space. It is also essential for the growth ofgranulation tissue. The principle growth factors that regulateangiogenesis are FGF, released by damaged endothelial cellsand macrophages, and vascular endothelial growth factor(VEGF) which is released by keratinocytes and macrophages[6].

The maturation phase usually begins 3 weeks after injuryand can take up to 2 years to complete [10]. Unlike uninjuredskin, the arrangement of newly formed collagen fibres in thewound is random and disorganised. The remodelling of col-lagen fibres into a more organised lattice structure graduallyincreases the tensile strength of the scar tissue, though thisnever exceeds 80 percent of the strength of intact skin. Re-modelling of the ECM involves a balance between collagensynthesis and degradation, which is operated by several en-zymes, like matrix metalloproteinases (MMPs), neutrophil-released elastase and gelatinase, collagenases and stromely-sins [11].

Wounds that do not heal within three months are con-sidered chronic. In acute wounds, there is a precise balancebetween production and degradation of molecules such ascollagen; in chronic wounds this balance is lost and degrada-tion plays too large a role. Chronic wound bed has been de-monstrated to differ from acute wounds for a higher concen-trations of proteases (such as MMPs) [12] and lower levelsof growth factors and cytokines [13]. A high and prolongedproteolytic activity may lead to the degradation of growthfactors, detaining the wound in the inflammatory stage fortoo long [14].

2. Nanotherapies for Wound Healing

An increasing number of products emerging from the appli-cation of nanotechnology to the science of wound healing iscurrently under clinical investigation. The current nanoscalestrategies, both carrier, drug related and scaffold (Figure 2),that target the main phases of wound repair will be discussed,highlighting the cellular signals involved (Table 1).

2.1. Nanoparticle-Bearing Endogenous Molecules. As wepointed out previously, soluble active proteins (cytokinesand growth factors) govern the progression of the healingphases through the modulation of the cellular and molecularcomponents involved [43]. Because of the control they exerton wound repair, bioactive proteins have gained progres-sive interest for the treatment of chronic dehiscent wounds.Clinical trials involving the application of exogenous recom-binant growth factors to chronic wounds have been con-ducted for the past 10 years in anattempt to find a way toaccelerate healing [44]. Although the results of several pilottrials have been encouraging, the overall results have beensomewhat discouraging. To date, only a single recombinant

growth factor—recombinant human PDGF-BB—has beenapproved by the United States Food and Drug Administra-tion, and only for use in diabetic foot ulcers [45]. In fact, themajor limitation to the topical use of growth substances isthat in human plasma the half-life of proteins involved insignalling cascades is very short (few seconds) due to closecontrol and inactivation by protease inhibitors [46]. There-fore, the clinical use of endogenous factors as pure com-pounds is limited by the breakdown by the proteolyticenzymes that enrich the wound site [47]. Several attemptshave been already made to provide biological moleculeslong-term protection from enzymatic degradation (liquids,gels, collagen sponges, and gene transfer). Recently, thedevelopment of nanoscale systems for drug delivery hasopened new possibilities to enhance the biological efficacy ofmolecules through a controlled release for extended periodsof time [48]. Endogenous active molecules that have been en-gineered nowadays include thrombin, nitric oxide, growthfactors, opioids, and protease inhibitors.

Thrombin (also termed activated factor II or factor IIa) isa protein involved in the final stage of the coagulation cascadeactivated by wounding. In addition to its well-documentedrole in the formation of fibrin clots and platelet activation,thrombin has direct effects on inflammatory cells, fibrob-lasts, and endothelial cells: it stimulates chemotaxis and ag-gregation of neutrophils, lymphocytes, and monocytes cells,and the proliferation of fibroblasts, epithelial and endothelialcells [49, 50]. Thus, thrombin may play an important rolein initiating early cellular events in tissue repair [49]. Since1988 researchers suggested that thrombin receptor-activatingpeptides could be useful in vivo to mimic the natural effectsof thrombin interaction with receptors on various types ofcells [51], a delivery systems that could provide thrombin along-term protection from its natural inhibitors (antithrom-bin and activated protein C) has been pursued. In recentyears, nanobiotechnology has provided the means to en-hance the bioavailability of thrombin by conjugation withiron oxide nanoparticles (γ-Fe2-O3 conjugation) [15]. Ani-mal models were tested for wound response to the treatmentwith conjugated thrombin: on a 28-day treated wound, re-sults obtained analysing tensile strength indicated a signi-ficant acceleration of healing process when comparedwith free-thrombin-treated wounds and untreated wounds.Obtaining a greater tensile strength may potentially reducesurgical complications such as wound dehiscence.

Nitric oxide (NO) is a small radical, formed from theamino acid L-arginine by three distinct isoforms of nitricoxide synthase. The inducible isoform (iNOS) is synthesizedin the early phase of wound healing by inflammatory cells,mainly macrophages. However, many cells participate in NOsynthesis during the proliferative phase after wounding. NOregulates collagen formation, cell proliferation, and woundcontraction in distinct ways in animal models of woundhealing [52]. NO is also a well-known antimicrobial agent,interfering directly with DNA replication and cell respirationthrough the inactivation of zinc metalloproteins [53]. Treat-ment of acute and chronic wound failure with NO has beenfor years a major unresolved goal. Attempts at novel nitric


Gelatin microparticle

Poly(lactide-co-glycolide)nanoparticle

Carboxymethyl chitosannanoparticle

Polyacrylate nanoparticle

Silica nanoparticle

Poly(lactic acid) nanoparticle

Hyaluronannanoparticle

Neutrophil-derivednanoparticle

Solid lipid nanoparticle

Embedded growth factors Embedded opioids Embedded DNAEmbedded

protease inhibitors

Lipoxin Aanalog

Resolvin D1

Embeddedantibiotic

Embedded

EGF PDGF

PDGF

nitric oxide

TGF-βAnti-VEGF

plasmid DNA

Inflammation Remodelling Wound contraction

2D/3Dnanofibrous sheets

Stem cells-seeded scaffolds

Conjugated thrombin Conjugated antibiotics

Silver nanoparticles

Silver-loaded nanofibers

VAN

VAN

VANN

H

O O THROO

Fe Fe

Em

bedd

edn

anop

arti

cles

Nan

osca

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dsC

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dn

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cles

Pu

ren

anop

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cles

Figure 2: Nanostrategies currently in use for promoting skin wound healing. In the main panel, nanostrategies currently in use to improvehealing are illustrated. The therapeutic potential of nanosphere-based strategies and nanoscaffolds is emphasized through correlation withthe healing phase to which the biological action is targeted. In the right panel, materials explored for nanoparticulate-delivery systems arelisted. Two- and three-dimensional nanofibrous sheets can be made of both degradable (collagen and chitosan) and nondegradable (PLA,PLACL, polyurethane, and PVA polymers) nanofibers.

oxide therapies, in the form of nitric oxide donors, haveshown limited potential in treating cutaneous infection [54].A novel approach is offered by recently engineered nanopor-ous materials, that make possible the storage and delivery ofsmall gaseous short-lived NO, allowing the free radicals toexert their antibacterial activity. Using silane hydrogel-basednanotechnology [23], NO remains trapped and stable withina dry matrix until the matrix is exposed to moisture. The drymatrix allows for NO nanoparticles to be easily stored andapplied. Once exposed to moisture, the drug is released fromthe nanoparticle over an extended period of time at a relative-ly fixed concentration. This sustained release distinguishesnanoparticles from other vehicles, such as injections, thatrelease a large concentration of the drug with a rapid

return to baseline. The ease of storage, application, andthe ability to alter release rate and concentration withminimal risk of toxicity make this powder formulationideal for cutaneous delivery. Authors showed promisingresults from NO-releasing nanodelivery systems in pre-venting and treating skin infections caused by methicillin-resistant Staphylococcus aureus (MRSA), which is oneof the major causes of hospital-acquired infections [55,56]. Moreover, NO-releasing silica nanomolecules havebeen demonstrated to exert a bactericidal activity againstPseudomonas aeruginosa [57] and Acinetobacter bauman-nii [58]: both microbial agents have become an increas-ingly prevalent cause of hospital-acquired infections duringthe last 15 years, the majority of clinical A. baumannii


Table 1: Executive summary table.

Inflammation

(i) thrombin is one of the first products of the coagulation cascade occurring during haemostasis, and is responsible for plateletactivation and aggregation, leading to the formation of the “platelet plug” and allowing cells and fluid to enter the wound bed. In humanplasma, thrombin is rapidly degraded (15 sec). In order to provide long-term protection, it has been conjugated with iron-oxidenanoparticles for treatment of incisional wounds in rats [15]

(ii) bacterial infection and sepsis exacerbate the inflammatory state and cause tissue damage

(a) nanoparticles bearing vancomycin or N-methyltiolated β lactams have been developed to act against wound contamination by MRSA[16–19]

(b) silver-based nanoparticles were developed to take advantage on the multilevel antibacterial action of silver and try to reduce thedevelopment of microbial resistance. Pure biostable nanoparticles were produced through photoassisted reduction and ion stabilization.Silver nanoparticles were also loaded into nanofibers [20]. A direct promotion of wound healing by silver nanoparticles throughreduction of the cytokine-modulated inflammation and cell migration and proliferation was also demonstrated [21, 22]

(c) donor NO silica nanoparticles showed speed healing by killing both Gram-positive and Gram-negative bacteria and overcoming theNO deficiency [23]. No-releasing nanoparticles may also potentially accelerate healing by a promotion of angiogenesis and tissueremodelling [24]

Proliferation

(i) the aim of growth factors is to promote cell migration into the wound site, stimulate the growth of epithelial cells and fibroblasts, startthe formation of new blood vessels, and profoundly influence the remodelling of the scar. To enhance the in vivo efficacy of growthfactors they have been incorporated into polymer nanocarriers to sustain release. PLA/PLGA/PEG/hyaluronan/gelatin nanoparticlesembedded with different growth factors have been successfully applied on skin wounds [25–31]

(ii) opioids have been recently indicated as factors promoting keratinocytes migration. Solid lipid nanoparticles were embedded withopioids, confirming the influence of these drugs on keratinocytes migration [32, 33]

(iii) nanofibrous scaffolds: electrospunnanofibers networks support cell adhesion, proliferation, and differentiation mimicking thefibrous architecture of the extracellular matrix. Both degradable (collagen and chitosan) and nondegradable (PLA, PVA, PLACL, andpolyurethane) fibers are used for 2D and 3D constructs [34–36]. Scaffolds were also engineered to contain growth factors-releasingnanoparticles enhancing wound repair [37]

Remodelling

(i) matrix metalloproteinase collagenolytic activity appears to be upregulated in chronic wounds. Protease inhibitors were loaded intohuman derived nanoparticles, showing a proresolving action and accelerated healing [38]

(ii) gene therapy: nonviral polymeric gene delivery systems offer increased protection from nuclease degradation, enhanced plasmidDNA uptake, and controlled dosing to sustain the duration of plasmid DNA administration. Gene delivery systems are formulated fromPLGA polymers, polysaccharides, and chitosan [39–41]

(iii) stem cells: cell-based therapies hold the potential to promote vascularization and tissue regeneration. The hVEGF gene was deliveredthrough biodegradable polymeric nanoparticles: treated stem cells showed the engraftment of the tissue [42]

isolates displaying high-level resistance to common antibi-otics [59]. Also, the beneficial effects of NO seem toovercome the antimicrobial activity and involve directly thehealing process. Experiments demonstrated that pharmaco-logical or genetic (NOS knockout) reduction of NO impairsthe speed and effectiveness of wound healing, and thatthis process can be reversed by restoring NO productionwith increased NOS substrate (arginine), or by transfectingwith the missing NOS gene [24]. Thus, application of NO-releasing nanoparticles may potentially accelerate healing notonly by killing bacteria but also by a promotion of angiogen-esis and tissue remodelling.

The clinical use of growth factors in wound healing hasbeen of great interest recently. They have the potential to ac-celerate the healing process by attracting cells into the woundsite (TGF-β), promoting cell migration, stimulating the pro-liferation of epithelial cells and fibroblasts (FGF and PDGF),as well as initiating the formation of new blood vessels (FGFand VEGF), and finally participating in the remodelling ofthe scar [46]. A mean of enhancing the in vivo efficacy ofgrowth factors, preventing degradation from proteolytic

enzymes, is to stabilize protein structure and biological ac-tivity, prolonging the length of time over which growth fac-tors are released at the delivery site [60, 61]. The period ofdrug release from a polymer matrix can be regulated by drugloading, type of polymer used and the processing conditions.Delivery systems have been designed in a variety of config-urations and have been fabricated from different types ofnatural and synthetic polymers, both degradable and non de-gradable. Poly(lactic acid) (PLA) and poly(lactic-co-glycolicacid) (PLGA) have been demonstrated to be biocompatibleand biodegradable suitable materials. To achieve long-termin vivo circulation, the surface is generally modified withpolyethylene glycol (PEG), reducing the clearance by the re-ticuloendothelial system [25]. In biodegradable carriers,growth factor release is controlled by the polymer matrix’srate of degradation, which causes changes in the morpho-logical characteristics of the materials, such as porosity andpermeability [26]. Particulate delivery systems explored in-volve TGF-β-embedded gelatine microparticles [27], EGF inPLA microspheres [28], FGF in gelatin microspheres [29],and PDGF-embedded PLGA nanospheres [30]. In general,


porous materials seem to retain a higher specific surface forthe adsorption and release of active components and en-hancement of drug release. Nevertheless, the particles showsome limits which are associated with the use of organic sol-vents in the production process, leading to pollution and tox-icity of the product [62]. Therefore, alternative techniques offabrications have been proposed, such as spray drying, whichis based on the use of supercritical fluids (especially CO2) andoffers the advantages of being environmentally safe and inex-pensive [63]. A notable recent application of this technologyfor the delivery of growth factors in vivo was offered by Zavanet al. [31]: hyaluronan-based (HYAFF11) porous nanoparti-cles were embedded with PDGF as a system designed for thein vivo treatment of skin ulcers. PDGF is known since 1986to successfully promote chronic wounds healing through astimulation of chemotaxis, proliferation and ECM deposi-tion [64]. HYAFF11, the benzyl ester of hyaluronic acid, isa biopolymer that has found numerous applications forin vitro reconstruction of skin as well as for the in vivo regen-eration of small arteries and veins [65]. HYAFF particles havethe ability to absorb growth factors and to release them in atemporally and spatially specific event-driven manner. Thistimed and localized release of PDGF promoted optimal tissuerepair and regeneration of full-thickness wounds.

Beside the dedicated growth factors, opioids have beenrecently indicated as factors promoting keratinocytes migra-tion [66]. This finding is of great interest because of a possibleenhancement of wound healing through topical applicationsof opioids at the wound site for pain reduction. Conventionalformulations failed to consistently provide sufficient paincontrol in patients, possibly due to local drug degradation orinsufficient concentrations at the target site [32]. Since longintervals for painful wound dressing changes are intended,the formulations should allow for prolonged opioid release.Bigliardi et al. [32] first developed nanoparticulate carriers toincrease opioids skin penetration and slow the release of theloaded drug. Experiments on human keratinocyte-derivedcell line HaCaT showed that opioids stimulated cell migra-tion and closure of experimental wounds. Enhancement ofmigration was concentration-dependent and could be block-ed by the opioid receptor antagonist naloxone, indicating aspecific opioid-receptor interaction. Kuchler followed [33]demonstrating that morphine-loaded solid lipid nanopar-ticles accelerated reepithelialisation on a human-based 3D-wound healing model. On standardised wounds, keratino-cytes almost completely covered the dermis equivalent after4 days, which was not the case when applying the unloadedparticles. In conclusion, acceleration of wound closure, lowcytotoxicity, and irritation as well as possible prolonged mor-phine release make solid lipid nanoparticles an interestingapproach for innovative wound management.

Matrix metallo proteinases (MMPs) are zinc-dependentendopeptidases that cleave most macromolecules within theECM during the maturation of the wound [67]. The processof remodelling constitutes a balance between collagen pro-duction, breakdown, and remodelling. The biological activ-ity of MMPs is strictly balanced by the presence of specific tis-sue inhibitors (TIMPs). A tightly coordinated expression ofspecific combinations of MMPs and TIMPs appears to be

necessary for proper wound maturation. As previously stat-ed, an uncontrolled proteolytic activity leads to delayed heal-ing through degradation of growth factors. In light of theseconsiderations, research approaches to improve the remod-elling phase are directed to manage enzymatic activity ofMMPs through the topical application of protease inhibitors.Norling et al. [38] recently took advantage of aspirin-trig-gered resolving D1 and lipoxin A4 analogs and developed acarrier providing stable biological activity to these naturalcompounds. Human-derived nanoparticles were enrichedwith the protease inhibitors, and wound healing reactionswere tested in a murine model. Polymorphonuclear cell in-flux showed a dramatical reduction in treated wounds, withshortened resolution intervals, and a proresolving action thatin the end accelerated healing.

2.2. Nanoparticle-Bearing Antibiotics. During the healingprocess, infection is an issue potentially compromising thefinal wound closure, exacerbating the tissue damage [68].Nowadays, wound infection is no longer the ominous eventas at the beginning of the 20th Century, when infection ofwounds, especially burns, was the major cause of morbidityand mortality (over 50%) [69]. Nevertheless, an appropriateantimicrobial therapy of the wound controlling colonizationand proliferation of microbial pathogens, including multi-drug-resistant organisms, is still required for an appropriatewound care. Staphylococcus aureusis one of the most com-mon pathogens involved in wound infections. The pharma-cological treatment encounters today severe limitations dueto the development of antibiotic resistant strains. For exam-ple, penicillinase (an enzyme that breaks down the β-lactamring of the penicillin molecule) is responsible for the resis-tance to penicillin of Staphylococcus aureus: the failed treat-ment of staphylococcal local infections leads to the onset ofserious late complications (bacteraemia, sepsis, toxic shocksyndrome) [69].

The delivery of antibiotic therapy via nanoparticles offersgreat potential advantages. Particularly, a controlled releasewould decrease the number of doses required to achieve thedesired clinical effect, potentially reducing the risk of devel-opment of antibiotic resistance. The physiochemical proper-ties of nanoparticulate drug delivery systems (size, surfacecharge, and nature) are determinant in vivo for the factorsdelivery. In particular, it is known that 20–200 nm particu-lates are suitable for delivery of therapeutics; larger size par-ticles suffer from quick uptake by the reticulo-endothelialsystem and rapid clearance from circulation, whereas thesmaller size will tend to cross the fenestration in the hepaticsinusoidal endothelium, leading to hepatic accumulation[70].

In the treatment of staphylococcal infections, the latestgeneration antibiotic and assumed to be the most useful atthe moment is vancomycin. Several vancomycin-modifiednanoparticles have been developed to enhance the pharma-cokinetics and pharmacodynamics of the antibiotic mole-cule. Hachicha et al. [16] recently proposed the use of vanco-mycin-conjugated nanoparticles for intraocular continu-ous release injection for endophthalmitis prophylaxis. Thedrug concentration was proved to be maintained above


the minimal inhibitory limit for 24 hours. Chakraborty et al.[17] developed carboxymethyl chitosan-based nanoparticles,which were loaded with vancomycin and proved effectiveagainst drug-resistant staphylococcal strains.

The effectiveness of nanobiotechnology in enhancing thetherapeutic properties of antibiotic molecules is emphasizedby the case of N-methylthiolated β-lactams: these com-pounds have been recently identified and proved effectiveagainst Staphylococcus bacteria, including MRSA. These lac-tams exert growth inhibitory effects on bacteria through amode of action that is distinctively different from other β-lactam antibiotics, with peculiar structure-activity patterns.Nevertheless, their potential application is limited by theexceeding low water solubility [18]. Thus, a methodologywas developed by Turos et al. [19] to obtain an emulsion ofpolyacrylate nanoparticles in which the drug monomer wasincorporated into the polymeric matrix: in vitro screensconfirmed the nanoparticles to be nontoxic to human dermalfibroblasts and stable in blood serum for 24 hours.

Within the bacterial species, resistance to all known anti-biotic classes has occurred due to mutation and horizontalgene transfer. This has led to anxiety regarding the futureavailability of effective chemotherapeutic options. Due to theoutbreak of infectious diseases caused by different patho-genic bacteria and the development of antibiotic resistance,the pharmaceutical companies and the researchers are nowsearching for new antibacterial agents.

2.3. Silver-Based Nanoparticles. The use of silver for the treat-ment of ulcers is reported since the 5th Century B.C. In the17th and 18th centuries, silver nitrate was already used forulcer treatment. The antimicrobial activity of silver was esta-blished in the 19th century. Nevertheless, after the introduc-tion of antibiotics in 1940 the use of silver salts decreased.Subsequently, silver salts and silver compounds have beenused in different biomedical fields, especially in burn treat-ment [71].

The antimicrobial activity of silver appears high: due toits multilevel (including multidrug resistance) antibacterialeffects [72] and low systemic toxicity [73], it provides an anti-bacterial effect that considerably reduce the chances of de-veloping resistance. Evidently, the greatest rate of silver ionsrelease is wished in order to enhance the clinical effect andavoid the insurgence of silver-resistant mutant bacteria.Nanotechnology has provided the means of producing purebiostable silver nanoparticles, either through photoassistedreduction and ion stabilization or loading of the metallic par-ticles into nanofibers [20]. In all cases, 7–20 nm silver nano-particles exhibited antibacterial (especially anti-Gram nega-tive) and antifungal activity, and they are also synergistic tocommon antibiotic therapy (streptomycin, kanamycin, andpolymyxyn B) [20]. Studies in vivo demonstrated also a directpromotion of wound healing by silver nanoparticles throughreduction of the cytokine-modulated inflammation: silver-induced neutrophils apoptosis, decreased MMPs activity, andnegative modulation on TGF-β resulted in an overall acceler-ation of wound healing rate and reduction on hypertrophicscarring [21]. Also, wound healing rate was demonstrated tobe increased by silver ions by a promotion of proliferation

and migration of keratinocytes along with a differentiationof fibroblasts into myofibroblasts, thereby promoting woundcontraction [22].

Though the progressive expansion of the therapeutic ap-plication of silver nanoparticles, the use of a metallic com-pound carries possible side effects that must be taken intoconsideration. Studies have been investigating the biosafetyof silver as a therapeutic agent, reporting an acceptable bio-compatibility [74], though the occasional development ofargyria (a cosmetic blue-grey coloration of the skin).

2.4. Nanoparticles and Gene Therapy. Polymeric gene deliv-ery systems offer several advantages for plasmid DNAdelivery, such as protection from degradation by the nucleaseand controlled prolonged release. The potential retained bythe modulation of gene expression in the process of woundhealing lead researchers to apply for an engineered systemfor DNA transfection. Actually, transfection capability hasbeen tested in vitro: biocompatible and biodegradable PLGApolymers [39] were engineered to obtain high plasmid load-ing efficiency [75] and then loaded withan antiangiogenicplasmid DNA (pFlt23k). The PLGA nanoparticles were pre-pared by a supercritical fluid extraction of emulsions basedon CO2: this allowed high loading of pDNA (19.7%, w/w),high loading efficiency (>98%), and low residual solvents(<50 ppm). The VEGF secretion by epithelial cells was signi-ficantly reduced, showing a potential value in treating wounddisorders in which VEGF is elevated.

In the course of studies on nonviral DNA carriers forgene delivery and therapy other materials have been applied,like polysaccharides and other cationic polymers. Chitosanalso was found to be particularly suitable, since it can pro-mote long-term release of incorporated drugs [76]. Masottiand Ortaggi recently described a nanofabrication methodthat may be useful for obtaining small DNA-containingchitosan nanospheres (38 ± 4 nm) for biomedical applica-tions [40]. Their reported osmosis-based method has generalapplicability to various synthetic or natural biopolymers, re-sulting in nanostructured systems of different size and shapethat may be used in several biotechnological applications.Chellat et al. also recently took advantage of the biochemicalproperties of chitosan to test DNA-loaded nanoparticles ona human macrophage cell line to study the potential modula-tion of the expression of proinflammatory cytokines, metal-loproteinases and their specific inhibitors [41]. The secretionof MMP-9 in cell supernatants increased significantly after 24and 48 h in comparison with nontreated cells. MMP-2 secre-tion was augmented only after 48 h with incubation of thehighest concentrations of nanoparticles (10 and 20 μg/mLDNA content). However, zymography studies showed thatsecreted MMPs were in their proactive form, while in thepresence of 10 and 20 μg/mL DNA-containing nanoparticles,the active form of MMP-9, but not MMP-2, was detected incell lysates. The results obtained were significative only forincreased secretion of metalloproteinases, possibly related tonanoparticles phagocytosis.

2.5. Nanoparticles and Stem Cells. Stem cells are lauded fortheir unique ability to develop into every kind of cell. Even if


in vivo studies and clinical trials have demonstrated limita-tions in reconstituting tissue due to the lack of microenviron-ment-control on proliferation and survival, the successfuluse of these cells for investigation towards disease therapy isstill pursued. Last decade has witnessed a growth in the fieldof nanoparticles technology for stem cells isolation, main-tenance, and regulation: nanoparticles and nano 3D archi-tectures have been developed to control stem cells prolifera-tion, differentiation, and maturation [77]. In terms of skinregeneration, VEGF high-expressing, transiently modifiedstem cells have been developed for the purpose of promot-ing angiogenesis [42]. In order to overcome the insufficientexpression of angiogenic factors and low cell viability aftertransplantation, nanotechnology has provided nonviral, bio-degradable polymeric nanoparticles to deliver hVEGF geneto human mesenchymal stem cells (hMSCs) and human em-bryonic stem cell-derived cells (hESdCs). Treated stem cellsdemonstrated markedly enhanced hVEGF production, cellviability, and engraftment into target tissues. Implantationof scaffolds seeded with VEGF-expressing stem cells (hMSCsand hESdCs) led from 2 to 4 fold higher vessel densities 2weeks after implantation, compared with control cells or cellstransfected with VEGF by using Lipofectamine 2000, a lead-ing commercial reagent. Four weeks after intramuscular in-jection into mouse ischemic hindlimbs, genetically modifiedhMSCs substantially enhanced angiogenesis and limb sal-vage, while reducing muscle degeneration and tissue fibrosis[42]. These results indicate that stem cells engineered withbiodegradable polymer nanoparticles may be therapeutictools for vascularizing tissue constructs and treating ischemicdisease.

2.6. Nanofibrous Scaffolds. The basic strategy of engineeredtissue regeneration is the construction of a biocompatiblescaffold that, in combination with living cells and/or bioac-tive molecules, replaces, regenerates, or repairs damagedtissues. The scaffold should possess suitable properties, likebiocompatibility, controlled porosity and permeability, and,additionally, support for cell attachment and proliferation.This artificial “dermal layer” needs to adhere to and integratewith the wound, which is not always successful for thecurrent artificial dermal analogues available. The high cost ofthese artificial dermal analogues also makes their applicationprohibitive both to surgeons and patients. Engineering der-mal substitutes with electrospun nanofibres have lately beenof prime importance for skin tissue regeneration. Simpleelectro spinning technology served to produce nanofibrousscaffolds morphologically and structurally similar to theextracellular matrix of native tissues. The engineered net-work has been shown to support cell adhesion, proliferation,and differentiation mimicking the fibrous architecture ofthe extracellular matrix [60]. The large surface area andporosity of electrospun nanofibers enables good permeabilityfor oxygen and water and the adsorption of liquids, and con-comitantly protects the wound from bacterial penetrationand dehydration. This feature shows electrospun nanofibersto be a suitable material for wound dressing, especially forchronic wounds such as diabetic ulcers or burns.

The electro spinning technique can provide both degrad-able (collagen; chitosan) and nondegradable (PLA, polyvinylalcohol (PVA) polymers) nanofibers for two-dimensionalnanofibrous sheets. Both sorts of biomaterials have beentested in vivo showing an increased rate of wound epithelia-lisation and dermis organization [34], as well as good anti-bacterial activity against the Gram-positive and Gram-nega-tive bacteria [35]. Nanofibrous scaffolds of poly(L-lacticacid)-co-poly(ε-caprolactone) (PLACL) and PLACL/gelatincomplexes were fabricated by Chandrasekaran et al. [36].These nanofibres were characterized by fiber morphology,membrane porosity, wettability, and chemical properties byFTIR analysis to culture human foreskin fibroblasts for skintissue engineering. The results showed that fibroblasts prolif-eration, morphology, and secretion of collagen were signif-icantly increased in plasma-treated PLACL/gelatin scaffoldscompared to PLACL nanofibrous scaffolds. The obtainedresults proved that the plasma-treated PLACL/gelatin nanofi-brous scaffold is a potential biocomposite material for skintissue regeneration.

Moreover, nanofibrous constructs can be obtained witha 3D profile, even though they scarcely support cells seedingbecause of their high porosity. Several strategies have beendeveloped to improve cell infiltration, showing promisingresults [79]. Chong et al. [80] proposed a cost-effective com-posite consisting of a nanofibrous scaffold directly electro-spun onto a polyurethane dressing (Tegaderm, 3 MMedical)—Tegaderm-nanofiber (TG-NF) construct—fordermal wound healing. Cell culture was performed on bothsides of the nanofibrous scaffold and tested for fibroblastadhesion and proliferation. Results obtained in this studysuggested that both the TG-NF construct and dual-sidedfibroblast-populated nanofiber construct achieved signifi-cant cell adhesion, growth, and proliferation. This was a suc-cessful first step for the nanofiber construct in establishingitself as a suitable three-dimensional scaffold for autogenousfibroblast populations and providing great potential in thetreatment of dermal wounds through layered application.

Steps towards an enhanced regenerative effect will be toprovide scaffolds a delivery system for drugs, growth factorsor cytokines that may further promote cell function and tis-sue regeneration [81]. Jin et al. already worked towards thisdirection engineering PLGA microspheres in nanofibrousscaffolds to control the release of PDGF in vivo [37]. PDGFconcentration was evaluated in a soft tissue wound repairmodel in the dorsa of rats. At 3, 7, 14, and 21 days after-implantation, the scaffold implants were harvested followedby assessments of cell penetration, vasculogenesis, and tissueneogenesis. Gene expression profiles using cDNA microar-rays were performed on the PDGF-releasing NFS. The per-centage of tissue invasion into microspheres-containingnanofibrous scaffolds at 7 days was higher in the PDGFgroups when compared to controls. Blood vessel number inthe groups containing either 2.5 or 25 μg PDGF was increasedabove those of other groups at 7d (P < 0.01). Results fromcDNA array showed that PDGF strongly enhanced in vivogene expression of the CXC chemokine family memberssuch as CXCL1, CXCL2, and CXCL5. Thus, sustained releaseof rhPDGF-BB, controlled by slow-releasing microspheres


associated with the nanofibrous scaffold delivery system,enhanced cell migration and angiogenesis in vivo, and may berelated to an induced expression of chemokine-related genes.This approach offers a technology to accurately controlgrowth factor release to promote soft tissue engineering invivo.

3. Conclusions

In recent years, nanomedicine has experienced a progressiveexpansion: its great potential has attracted considerableinvestments from governments and industry, which are pre-dicted to increase in the near future, leading to further en-hancement and development. Similarly, the knowledge of thecellular and molecular processes underlying wound healinghas reached a level which let researchers apply for new thera-peutic approaches that act directly on cellular and subcell-ular events during the healing process. Nanotechnologytoday offers the means to overcome the dimensional barrierof currently used therapies for wounds and ulcers, toreach a dysfunctional molecular target and exert the thera-peutic action straight at the origin of the chronic condition.Nanocarriers possess an enormous potential, as nanopar-ticles-based delivery systems can be highly beneficial toaugment the therapeutic power of biological and syntheticmolecules. However the promising results brought by newtechnologies in therapeutics, the real biological effects ofnanoparticles have to be carefully assessed before introducingtheir use in clinical practice. In particular, the release of activepeptides may possibly cause interferences with some biolog-ical functions and cellular processes. With regard to this, theincorporation of a targeting ligand into nanoparticles hasbeen proposed, in order to give them site-specificity; also, awide variety of biomaterials has been prompted to meet thespecific biological requirements [82]. This could be especiallycritical for tissue regeneration, where the biomaterial prop-erties could augment the reparative process, or hinder it dueto undesirable attributes of the material.

Despite its recognized importance, there have not beensystemic studies that probe the targeting efficiency of nano-particles nor international standards on their toxicology andbiocompatibility. Our wish is that further research is to beextended to the applied science of nanotechnology. The re-volutionary potential offered by nanoscale therapeutics appl-ied to wound healing involves the need to develop interna-tional standards on their biocompatibility. A safe and target-ed use is required to prove beneficial to such a world-wideissue, which is chronic wounds and ulcers care.

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